1. Field of the Invention
This invention relates to pluripotent parenchymal hepatocytes derived from the epiblast of pig blastocyst which have been cultured continuously for a number of years, indicating an infinitely self-renewing cell population. The stem cell characteristics of the hepatocytes indicate that the cells are unique for investigations of liver differentiation and organogenesis and are particularly desireable for use in artificial liver devices.
2. Description of the Relevant Art
Liver transplantation has become widely accepted as an effective treatment for chronic and acute liver disease. One of the major problems associated with the transplantation process, however, as been the need for an effective means for providing temporary support for patients awaiting an available donor organ. Extracorporeal devices for providing heart, kidney and lung support are well-established, however, the development of such devices which are effective for liver support has proven more elusive.
Two basic approaches have emerged as promising. Both utilize a device such as a hollow fiber cartridge containing liver-derived cells which acts as a perfusion apparatus. Primary hepatocytes attached to microcarriers (Rozga et al. 1993. Hepatology. vol. 17(2), pp. 258-265; Rozga et al. 1994. Ann. Surg. vol. 217(5), pp. 502-511) represents one approach, while transformed cells such as those derived from hepatoblastoma cell lines (Sussman et al. 1992. Hepatology. vol. 16(1), pp. 60-65) are another. These approaches have several drawbacks, however. For example, primary cells have a very limited culture capability, therefore there is a requirement for continuous cell preparation. Transformed cells, on the other hand, grow well in culture, but, since they are transformed, they exhibit functional characteristics which may be different from normal cells. Both also suffer from the lack of accessory cells such as epithelial and bile-duct epithelial cells which are important in the function of a normal liver. The need thus exists for the discovery of normal hepatocytes capable of long-term culture which could effectively mimic liver function in an extracorporal device.
The continuous secondary culture of normal parenchymal hepatocytes has previously been unsuccessful, (Williams et al. 1977. In Vitro. vol. 13, pp. 809-817; Guguen-Guillouzo et al. 1983. Exp. Cell Res. vol 143, pp. 47-54; Perraud et al. 1991. Exp. Cell Res. vol 195, pp. 59-65). In vitro hepatocyte studies have largely been limited to the use of primary cultures of hepatocytes established after collagenase perfusion of rat or mouse livers (Williams et al., supra; Berry and Friend. 1969. J. Cell Biol. vol. 43, pp. 506-520). These techniques were also amenable to the establishement of primary hepatocyte cultures in non-rodent species such as the pig (Caperna et al. 1985. J. Anim. Sci. vol. 61, pp. 1576-1586). However, the use of primary cultures is problematic since it is labor intensive and inherently variable. In addition, other epithelial cells, fibroblasts and macrophages were common "contaminants" in primary liver cell culture, and these cells can quickly overgrow or otherwise interfere not only with maintaining cultures but also with experimental manipulations and measurements (Caperna et al., supra; Furukawa et al. 1987. In Vitro. vol. 23, pp. 339-348; Langenbach et al. 1979. Cancer Res. vol 39, pp. 3509-3514). Moreover, normal liver parenchymal cell activities, such as .alpha.-fetoprotein (AFP) and albumin production, and various enzymatic functions were often lost within one to two weeks of establishment in primary culture (Guguen-Guillouzo et al., supra; Langenbach et al., supra).
An alternative to normal parenchymal hepatocyte cell culture has been the establishment of numerous human and rodent hepatocarcinoma-derived cell lines (Richardson et al. 1969. J. Cell Biol. vol. 40, pp. 236-247; Aden et al. 1970. Nature (London). vol. 282, pp. 615-616). Although several of these hepatoma cell lines were minimally deviated and expressed various proteins representative of normal hepatocyte function, they were likely to be abnormal in several aspects, particularly growth control. For example, some of these cell lines were highly tumorigenic when placed in vivo (Richardson, supra; Knowles et al. 1980. Science. vol. 209, pp. 497-499). Also, while hepatocyte growth factor (HGF) is mitogenic for primary hepatocytes, it has been reported to be cytostatic for the hepatoma cell lines HepG2, Hep3B and H35 (Higashi and Shima, 1993). Some human hepatoma cell lines also chronically produced hepatitis proteins (Aden et al., supra; Knowles et al., supra). Thus, hepatoma-derived cell cultures may confuse assessments of normal parenchymal biology in vitro, and in vivo assessments may not be possible. The ability to sustainably culture normal, functional parenchymal hepatocytes in secondary culture is therefore novel and of great value for the routine study of hepatocyte biology.
It has been hypothesized that the liver contains stem cells which support its general long term function and its regenerative ability (Sigal et al. 1992. M. J. Physiol. vol. 263, p. G139; Fausto and Meade. 1989. Lab. Invest. vol. 60, pp. 4-13). Stem cells have not been identified, although embryonic phenotypic gene expression, e.g. AFP, was found in some proliferating liver epithelia following the administration of hepatotoxic agents (Fausto et al. Purification and culture of oval cells from rat liver. In Pretlow and Pretlow, eds. Cell Separation: Methods and Selected Applications, Vol. 4. Orlando: Academic Press, 45-77; Evarts et al. 1987. Carcinogenesis. vol. 8, pp. 1737-1740). Liver "oval" cells and intrahepatic bile duct cells were also proposed as candidate liver stem cells or facultative stem cells (Sigal et al., supra; Fausto and Meade, supra). A consensus of data indicated that liver stem cells would express AFP and albumin and would be capable of differentiating into bile duct cells and parenchymal hepatocytes (Shiofiri, N. 1981. J. Embryol. Exp. Morphol. vol. 79, pp. 149-152; Evarts et al. 1989. Cancer Res. vol. 49, pp. 1541-1547; Germain et al. 1988. Cancer Res. vol. 48, pp. 4909-4918).
Thus although attempts have been made to establish normal hepatocyte cell lines which are capable of culturing over a long period of time (i.e., years) without losing characteristic activity, accomplishment of this goal has heretofore been unsuccessful.